The present invention relates to improved methods, compositions and kits for the detection of RNA molecules. More particularly, the invention relates to the use of base-modified, duplex-stabilizing nucleoside triphosphates in reverse transcription reactions for improving duplex stability during subsequent amplification-based detection of short RNA sequences, such as miRNA.
Micro RNAs (miRNAs) are a highly conserved class of small RNA molecules that are transcribed from DNA but are not translated into protein. miRNAs are processed into single stranded ˜17-24 nucleotide (nt) molecules that become incorporated into the RNA-induced silencing complex (RISC) and have been identified as key regulators of development, cell proliferation, apoptosis and differentiation. RISC mediates down-regulation of gene expression through translational inhibition, transcript cleavage, or both. RISC is also implicated in transcriptional silencing in the nucleus of a wide range of eukaryotes.
miRNAs and other small RNA molecules have been implicated in a number of human diseases such as cancer, cardiovascular disease, viral infection and metabolic disorders. Accordingly, it is critical that specific and sensitive analytical methods are available for detecting when, where and at what levels small RNAs are expressed (whether up or down regulated), in order to realize the full diagnostic and therapeutic potential of this important new class of targets. Unfortunately, due to their small size, applying conventional nucleic acid detection methodologies, such as amplification-based techniques, to the detection of small RNAs has been problematic. Their small size offers little sequence for designing hybridization probes and primers and, in fact, most conventional PCR primers are similar in length to the miRNAs themselves.
Various approaches have been used for detecting miRNAs including Northern blots, primer extension, signal-amplifying ribozymes and some amplification-based techniques. However, these and other conventional strategies for detecting small RNAs have been associated with problems relating to specificity, sensitivity, expense and/or ease of implementation.
Therefore, there is a need in the art for versatile, simple, and inexpensive compositions, methods and kits for use in the detection of small RNA sequences, which allow for rapid and robust amplification and detection. The present invention addresses these needs and offers other related advantages.